As for qualitative or quantitative measurement of a target substance in biological sample, technique employing the substance capable of competing or specifically binding with said target substance, with which a labelling agent having particular signal is bonded, has been so far known widely. Particularly, the assay based on the antigen-antibody reaction or hybridization of nucleic acid is very useful, since the assay permits measurement of said target substance with high sensitivity.
These assays permit to measure the presence or absence of the target substance or either its amount or concentration by binding a substance, to which the labelling agent had been bonded, directly or indirectly with the target substance, and by competing a substance or its derivative, to which the labelling agent had been bonded, with the target substance, followed by measuring signal intensity emitted from the label.
As one of the labelling agent for such uses, radioactive isotopes have been known and their uses have permitted to detect the target substance with high sensitivity. Radioactive isotopes, however, possess a large disadvantage that their storage, use, and disposition, accompany danger, so that in recent years, the use of non-radioactive labelling agents instead of radioactive isotopes has increased rapidly.
As a typical non-radioactive labelling agent, enzyme has been known. Particularly, the method employing enzymes as the labelling agent has found wide use in immunoassay. Some significant problems, however, exist in the use of enzymes as the labelling agents.
Namely, it can be cited that reproducibility of the results of assay using enzyme as the labelling agent are low due to the fact that enzyme is readily affected by several conditions such as temperature, sample's characteristics, that the enzymes commercially available are generally expensive, etc.. It can be also cited that, in the immunoassay, enzyme is bonded with the specific binding agent which binds specifically or competes with the target substance, but, as a fatal fault, the activities of both enzyme and specific binding agent are reduced as a result of bonding the enzyme to the specific binding agent.
Fluorescent substance is a one of the candidates of a non-radioactive labelling agent other than enzyme. As fluorescent substances so far employed as the labelling agent, fluorescein, rhodamine, dansyl chloride, umbelliferone, etc. are known.
The measurement method using fluorescent substance as the labelling agent takes advantage of fluorescence phenomenon, i.e. the phenomenon that certain compound, when receiving certain exciting light, emits proper light based on the electronic configuration of said compound, and in principle high sensitivity can be expected in this method. The use of fluorescent substance as the labelling agent, however, raises some problems.
One of said problems is the high background noise caused by Rayleigh scattering. Furthermore, as for measurement of the target substance in biological samples such as serum and urine, another problem is that a number of fluorescences derived from substances in biological samples seriously disturb detection of the fluorescence derived from said labelling agent.
These problems are primarily due to the fact that fluorescent substances so far employed generally have small Stokes shifts, and the particular property to distinguish the fluorescence derived from the labelling agent from the background fluorescence does not exist. Providing that such problems are dissolved, use of fluorescent substance as the labelling agent will become a very useful means in the field of analysis.
It has been found in recent years that complex formed from certain ligand and rare-earth metal ion emits strong fluorescence, wherein exciting light is absorbed by the ligand and the energy is transferred from the excited triplet state of the ligand to the rare-earth metal ion, so that fluorescence based on transition of the f orbital electron of said rare-earth metal ion is observed. As rare-earth metal, europium, terbium, samarium, etc., are exemplified, these rare-earth metals themselves possess intrinsic fluorescences.
In such complex, exciting wavelength depends upon the kind of ligand and emission wavelength depends upon the kind of rare-earth metal. And, such fluorescence derived from complex have at least 100 nm or more of Stokes shift and also possesses much longer fluorescence lifetime of the order above 1 .mu.s compared with the background fluorescences derived from protein and others with fluorescence lifetime of about 10 ns. Accordingly, taking these advantages, it is possible to distinguish the fluorescence derived from said complex completely from other background fluorescences, so that the fluorescent complex bearing rare-earth metal ion becomes favorable as the labelling agent.
Some of the compounds forming complex with rare-earth metal ion are well known already.
As one of these, .beta.-diketone forming complex with europium ion or samarium ion can be cited.
As regards the complex, various properties are reported, including that .beta.-diketone is a divalent ligand and more than 3 molecules or more of this ligand usually coordinate with europium ion (Krishna C. Joshi et al., Journal of Fluorine Chemistry, 13, 261-265 (1979) ; Livingstone S. E. et al., Aust. J. Chem., 29, 1,845-1,850 (1976); H. Hang et al., Nippon Kagaku Kaishi, 1, 66-73 (1981), etc.)
As an example employing such complex as the labelling agent, a description as to the antibody, to which the complex formed from .beta.-diketone and europium ion or terubium ion is bonded, is found in Japanese Patent Publication (1987) 62-18868.
However the stability of the complex of .beta.-diketone and a rare-earth metal ion is so low that, under practical assay conditions, the complex can not be maintained throughout the assay period. So it is very difficult to use the complex as the labelling agent in the immunoassay and others.
A number of reports as to the complex formed from rare-earth metal ion and ethylenediamine tetraacetic acid (EDTA) including that by Nakatani H. et al. (The Review of Physical Chemistry of Japan, 42, 103-107 (1972)) are also found.
As an example using such complex as the labelling agent, a description as to EDTA derivative which coordinates lanthanide ion strongly and has the functional group, being capable of binding the complex to antigen or antibody, is found in Japanese Patent Application Kokai (1985) 60-500767.
Complex of EDTA and rare-earth metal ion has the excellent stability of the complex itself, but has a fatal fault that fluorescence enhancing property due to the complex formation is lacking. Namely, EDTA lacks the ability to absorb and transfer energy and the fluorescence intensity derived from the complex of EDTA and said rare-earth metal ion is very small. So, the complex of EDTA and rare-earth metal ion has a similar fault in the use of the preceding fluorescent substance so far employed.
In Japanese Patent Application Kokai (1982) 57-186170, E. Soini, Hemmile, and others revealed a method enhancing the fluorescence intensity by allowing metal ion to transfer between two kinds of complex-forming compound consisting of non-fluorescent and fluorescent compounds. Concretely, the complex of EDTA analogue and metal ion, which is highly stable but non-fluorescent, is employed as the labelling agent in specific binding reaction. After the reaction, detergent and complex-forming compounds are added to transfer the metal ion from the complex of EDTA analogue to the complex-forming compounds, resulting the formation of new fluorescent complex in liquid phase.
The crucial fault of the method by Soini, Hemmile, and others lies in a high possibility that, due to the transfer of metal ion between two kinds of complex-forming compound, another metal ion expected not to participate in this reaction system participates in the reaction system, i.e. that contamination takes place. Accordingly, the close attention must be paid in measurement not only to reagent and equipment but also to all apparatuses to be employed and experimental environment, in order to prevent contamination by metal ion from the outside.
As more improved fluorescent compound, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid was reported by Diamandis E. P. and others (Clin. Chem., 33, 2000 (1987); Journal of Immunological Methods, 112, 43 (1988)). In Japanese Patent Application Kokai (1989) 64-47952, a method for bonding the bovine serum albumin (BSA) labelled with the fluorescent compound to antibody and a method for binding, via avidin-biotin reaction, the avidin labelled with said fluorescent compound to the antibody labelled with biotin, i.e. method with the intervention of intermediate, were described.
The feature of said fluorescent compound by Diamandis and others lies in that the compound coordinates rare-earth metal ion at the nitrogen atom site of the phenanthroline ring and forms the bond with the amino group of protein by its sulfonylchloride group. The stability of the complex formed from the compound and rare-earth metal ion is lower than expected. Furthermore, the compound is not designed so as to be insensitive to aqueous quenching of fluorescence, which often encountered in the measurement of fluorescence in aqueous solvents, although this is understandable considering the steric structure.
Hence, in the use of the fluorescent compound, such problems result, as that, throughout or after the immunological reaction, a large excess of metal ion must be added and that the solid phase to which an antibody is immobilized must be dried before measurement of fluorescence. Furthermore, another problem results that the fluorescence intensity emitted by the complex formed from the fluorescent compound and rare-earth metal ion is not so strong as expected, so that, as was indicated in preceding Japanese Patent Application Kokai (1989) 64-47952, the sensitivity is not high enough for the practical use, unless proteins such as BSA and avidin intervene between the antibody as specific binding agent and the fluorescent compound to increase the number of label per an antibody molecule.
Alpha B. et al. and Blasse G. reported on the fluorescence emitted by the complex formed from cryptand and rare-earth metal ion (Angew. Chem. Int. Ed. Engl., 26, 266-267 (1987); Chemistry of Materials, 1, 294-301 (1989)).
Cryptand is comparatively favorable for the stability and the fluorescent property when used as complex. It has a problem, however, that introduction of a functional group for bonding to protein into its parent structure is difficult. Another problem is arisen from the fact that the process of said complex formation, i.e. chelating process, requires the reaction at high temperature for a prolonged time, although once metal ion is incorporated into the center of the basic structure of cryptand, the formed complex becomes stable.
As the large-ring compound capable of forming chelate, other than the well known preceding compound, compounds such as crown ether and large-ring polyamine are known.
Although these compounds with large-ring structure have the possibility to form comparatively stable complex with metal ion, they require that the cavity size of the ring must fit the ion radius of the metal to coordinate, so that possible combinations of the metal ion with the compound having ring structure are remarkably limited. It is supposed further that the complex of bipyridine, large-ring polyamine, or others is less stable than the complex using, as ligand, EDTA or others permitting the three-dimensional coordination, since the former having a comparatively planar structure can coordinate the metal ion only two-dimensionally.
As described above, the utilization of fluorescent substance as the labelling agent for measurement of the target substance in biological sample is considered to be very useful, although accompanied with subjects to be solved.
The solution of aforementioned subjects is achieved by the development of the fluorescent substance, which is stable, has a large Stokes shift and a long fluorescence lifetime, and is very liable to quenching even in aqueous solvent.